Random access system and method for polymerase chain reaction testing

ABSTRACT

A random access, high-throughput system and method for preparing a biological sample for polymerase chain reaction (PCR) testing are disclosed. The system includes a nucleic acid isolation/purification apparatus and a PCR apparatus. The nucleic acid isolation/purification apparatus magnetically captures nucleic acid (NA) solids from the biological sample and then suspends the NA in elution buffer solution. The PCR testing apparatus provides multiple cycles of the denaturing, annealing, and elongating thermal cycles. More particularly, the PCR testing apparatus includes a multi-vessel thermal cycler array that has a plurality of single-vessel thermal cyclers that is each individually-thermally-controllable so that adjacent single-vessel thermal cyclers can be heated or cooled to different temperatures corresponding to the different thermal cycles of the respective PCR testing process.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

(Not Applicable)

BACKGROUND OF THE INVENTION

The present invention relates to a system and method for isolating andpurifying a biological sample and for testing the biological sample,and, more particularly, to a high-throughput, random access system andmethod for isolating and purifying nucleic acid contained in abiological sample and for testing the biological sample, such as bypolymerase chain reaction testing.

Polymerase chain reaction (PCR) testing is a universally-accepted andwidely-practiced laboratory method for replicating or amplifying theconcentration of nucleic acid (NA), such as DNA, in a test tube.Replication/amplification takes place in an aqueous solution containinga concentration of DNA molecules. Pre-determined amounts of thepolymerase enzyme, oligonucleotide primers, tri-phosphates of the fournucleic acids or substrates, activators, and stabilizers are then addedto the aqueous solution, which is then subject to three thermal cycles,referred to as the denaturing cycle, the annealing cycle, and theelongation cycle.

During the first, denaturing cycle, the DNA double helix in aqueoussolution is melted between about 90 and 95 degrees Centigrade (° C.) sothat each strand of the double helix is separated from the other. Duringthe second, annealing cycle, the denatured aqueous solution is cooled toa temperature between about 50 and about 65° C., causing theoligonucleotide primers to attach to complementary nucleotide sequencesof each denatured DNA strand. Finally, during the elongation cycle, DNAdouble helixes are re-formed by elongation of the primers at atemperature between about 70 and about 72° C. More specifically, athermostable polymerase, such as Taq-polymerase, bonds nucleotides tothe primer templates attached to the complementary nucleotide sequences,which forms two new DNA double helixes where before there was just one.Accordingly, with every complete cycle, there is a doubling of thenumber of DNA molecules, so, the number of DNA molecules after n cyclesis equal to 2^(n).

The duration of each of the three thermal cycles is very brief andtypically measured in seconds. For example, the DNA molecules meltinstantaneously at about 95° C. during the denaturing cycle. If theprimers are available in sufficient concentration, primer hybridizationduring the annealing cycle only requires about one (1) or two (2)seconds. Finally, re-formation during the elongation cycle can occur ata bonding rate of about 80 per second. Hence, the elongation cycle needsonly about two (2) seconds. Thus, theoretically, each PCR cycle requiresabout five (5) seconds to complete.

In practice, however, the duration of each thermal cycle depends on therate of heat transfer, to heat or cool the aqueous solution at thepre-determined thermal cycle temperature. Variables that can affect theheating/cooling rates include, inter alia, the volume of the solution,the concentration of the aqueous solution, the thermal conductivity ofthe vessel holding the NA in aqueous solution, the thermal conductivityof the apparatus holding the vessel, and the method of applying andremoving heat, e.g., by conduction or convection.

Conventionally, PCR testing is performed in “batches”. For example,typically, a thermal cycling device, such as a PCR plate, holds 96vessels in a closely-spaced, 8×12 vessel-well pattern. Batch processingand large thermal cycling devices used for batch processing, however,have several shortcomings.

First, testing is not begun until each well in the thermal cyclingdevice is filled with a vessel, which adds time. Second, because thepre-determined temperatures for the annealing and elongation thermalcycles vary depending on the nucleic acid being tested for, such as HIV,HCV, HDB, and so forth, testing is not begun until each well in thethermal cycling device is filled with a vessel containing an aqueoussolution having a concentration of the same DNA molecule, which addseven more time. Third, because the vessels and thermal cycling deviceare introduced at once as a unit, the amount of time to bring thevessels and thermal cycling device to the pre-determined temperatureassociated with the thermal cycle will be greater than the amount oftime to bring an individual vessel to the pre-determined temperatureassociated with the thermal cycle.

These shortcomings of batch processing can be addressed by a system andmethod that provide random access to each of the thermal cycles andthat, moreover, provide individual temperature control over each vesselcontaining an aqueous solution.

U.S. Pat. No. 6,558,947 to Lund, et al. discloses a batch-type, thermalcycling device that enables one to control the temperature of eachvessel well in the device independently of the temperature of adjacentvessel wells. This, however, only addresses half the problem becausetime is still spent filling up each of the 96 vessel wells before thethermal cycling device is batched.

Therefore, it would be desirable to provide a high-throughput system andtesting method that provide random access to each of the thermal cyclesand that provide individual temperature control over each vesselcontaining an aqueous solution.

SUMMARY OF THE INVENTION

A system and method for preparing a biological sample having an initialconcentration of a nucleic acid (NA) for polymerase chain reaction (PCR)testing are disclosed. The system includes a nucleic acidisolation/purification apparatus and a PCR testing apparatus. Thenucleic acid isolation/purification apparatus magnetically captures NAsolids from the biological sample and then suspends the NA in elutionbuffer solution. The PCR testing apparatus subjects the eluted solutionto multiple cycles of the denaturing, annealing, and elongating thermalcycles associated with PCR testing.

More specifically, the PCR testing apparatus includes a multi-vesselthermal cycler array that has a plurality of single-vessel thermalcyclers that are each individually-thermally controllable so thatadjacent single-vessel thermal cyclers can be heated or cooled todifferent temperatures corresponding to the different thermal cycles ofthe respective PCR testing process.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the followingmore detailed description and accompanying drawings where like referencenumbers refer to like parts:

FIG. 1 shows a schematic of a random access system for PCR testing inaccordance with the presently disclosed invention;

FIG. 2 shows the nucleic acid isolation/purification apparatus of therandom access system for PCR testing in accordance with the presentlydisclosed invention;

FIG. 3 shows the PCR testing apparatus of the random access system inaccordance with the presently disclosed invention;

FIG. 4 shows a single-vessel thermal cycler for individually heatingcuvettes containing reaction liquid disposed in an amplification anddetection device in accordance with the presently disclosed invention;and

FIG. 5 shows a block diagram of a fluorimeter and the amplification anddetection device in accordance with the presently disclosed invention.

DETAILED DESCRIPTION OF THE INVENTION

The presently disclosed invention provides a random access system and amethod using the system for isolating and purifying nucleic acid (NA)contained in a biological sample and for testing the biological sample,such as by polymerase chain reaction (PCR) testing. PCR testing iswell-known to the art and will not be described in detail. Likewise, the“Boom patent” technique for isolating and purifying nucleic acid usingmagnetic particles referred to in this specification is the subject ofU.S. Pat. No. 5,234,809 to Boom, et al., which is incorporated herein inits entirety by reference.

Referring to FIGS. 1-3, a random access system 10 for isolating andpurifying NA contained in a biological sample and for testing theisolated and purified NA is shown. The system 10 provides highthroughput and continuous testing of aqueous samples, i.e., “reactionliquids”, containing some concentration of NA. NA is used herein torefer to single-stranded nucleic acid, i.e., RNA, and double-strandednucleic acid, i.e., DNA.

The system 10 (FIG. 1) includes a nucleic acid (NA)isolation/purification apparatus 20 (FIG. 2) and a PCR testing apparatus30 (FIG. 3), which are discussed separately below.

The Nucleic Acid Isolation/Purification Apparatus

The Boom patent discloses means for isolating and purifying NA in abiological material that initially contains some endogenous NA and/orsome foreign NA. The Boom process is rapid, reproducible, and simple andthe purified end product is undamaged and usable in other molecularbiological reactions, such as PCR.

The Boom process begins by mixing the NA-carrying biological material,such as blood, serum, semen, urine, feces, saliva, tissue cultures, cellcultures, and so forth, with large amounts of a chaotropic substance inthe presence of fine-grained silica particles. The chaotropic substancecauses the biological material to lose all of its NA. The loosened NA isbound to the silica particles.

Chaotropic substances can include, without limitation, guanidinehydrochloride, guanidine (iso)thiocyanate, potassium iodide, sodiumiodide, and the like. The NA-binding, solid-phase, silica particles caninclude glass powder, silicon oxide, silicon dioxide crystals, amorphoussilicon oxide, and the like.

The solid, silica-NA complexes are than separated from, e.g.,centrifically using a vortex, the aqueous solution. The aqueous solutionis discarded. The solids are again washed with a chaotropic buffer andagain subject to separation from the aqueous wash. This washing andseparation process can be repeated multiple times.

The solid, silica-NA complexes are finally washed using a water-alcoholsolution and acetone. The solution is dried to remove the acetone,leaving the solid, silica-NA complexes. The solid, silica-NA complexesare finally eluted with an aqueous elution buffer and the purified NA insolution, i.e., the “eluted solution”, is recovered.

The NA isolation/purification apparatus 20 of the presently disclosedinvention enables the practice of a method that generally follows theteachings of the Boom patent in isolating and purifying existing NA in abiological sample. Referring to FIGS. 1 and 2, samples containing aknown or unknown concentration of NA are removed from a sample handler21, such as used in the Advia Centaur® immunoassay system manufacturedby the Siemens Medical Solutions Diagnostics of Tarrytown, N.Y., anddispensed in tubes in a vortex/incubation device 22. The circularvortex/incubation device 22 includes forty-five (45) positions to holdtubes and is capable of rotating at about 1100 revolutions per minute(RPM). NA samples are inserted and removed from the vortex/incubationdevice 22 on each cycle. Advantageously, unlike conventional batchprocessing, samples containing a different NA, such as HIV, HCV, HDB,and so forth, can be inserted randomly into the vortex/incubation device22.

Those skilled in the art can appreciate that the number of positions inthe circular vortex/incubation device 22 is a function of many things,including desired throughput and time. Therefore, use of a circularvortex/incubation device 22 having more or fewer positions than 45 iswithin the scope and spirit of this disclosure.

An enzyme for degrading the protein structure of the NA, such asproteinase K (PK), is added to the tube containing an NA sample and themixture is incubated in the vortex/incubation device 22 at a temperatureof about 56° C. for about five (5) minutes. A lysis buffer, such as S9,magnetic silica particles (MP), carrier RNA, and an Internal Control(IC) are then mixed in with the NA sample. Once the proteinase K enzymedegrades the protein structure of the NA sample, the lysis bufferreleases the NA.

This mixture is then incubated and agitated in the vortex/incubationdevice 22 at about 56° C. for about fifteen (15) minutes. Incubation andagitation break down the NA-carrying sample, causing release of the NAfrom the sample. The released NA is then captured by the magnetic silicaparticles (MP). Alternative means of agitation can include, for example,ultrasound vibrations.

At the completion of each incubation and agitation cycle, the tubescarrying the NA sample are continuously and sequentially removed fromthe vortex/incubation device 22 and disposed on a sample tube conveyer24, such as a conveyor belt having appropriately dimensionedreceptacles. For example, tubes can be raised from the vortex/incubationdevice 22 to the tube conveyor 24 using a pneumatically-activatedelevator (not shown).

While on the tube conveyor 24, the liquid lysis buffer, i.e., S9, andsupernatant are separated, e.g., by aspiration, from the magnetic silicaparticles (MP) and the magnetic silica particles (MP) are washed. Eachwash cycle includes dispensing an aqueous wash solution into the tube;agitating the tube to re-suspend the magnetic silica particles (MP);performing magnetic separation on the contents of the tube; andaspirating and discarding the aqueous solution. Four (4) washing cyclesare an acceptable number; however, more or fewer washing cycles can beused, realizing that there may be greater NA isolation and purificationfrom more washing cycles and less NA isolation and purification fromfewer washing cycles.

Use of magnetic silica particles enables magnetic separation of thecaptured NA from the liquid lysis buffer and the supernatant. Aspreviously mentioned, the solid, very fine-grained, magnetic silicaparticles capture the NA. After a washing solution is added to the tube,e.g., using a syringe pump, and the tube is agitated, re-suspending theNA-carrying, magnetic silica particles, a magnetic field is applied tothe tube. In one embodiment, the magnetic field results from one or moremagnets disposed adjacent select positions along the path of the tubeconveyor 24. The magnetic field quickly attracts the NA-carrying,magnetic silica particles to one interior surface of the respectivetube.

Tube agitation is accomplished on and by the conveyor belt 24. Forexample, a solenoid (not shown) can be mechanically-coupled to theconveyor belt's 24 drive pulley (not shown), such as a two-part,spring-loaded drive pulley. When the solenoid is activated, it causesthe drive pulley and the conveyor belt 24 to vibrate. Alternatively, adrive motor (not shown) can be stepped back and forth at apre-determined frequency, which will cause the conveyor belt 24 toshake.

After the final wash cycle, an elution buffer (E) is added to theNA-carrying, magnetic silica particles and the tube containing theeluted sample is transferred to another incubation/agitation ring 26,where the analyte-enriched NA is separated from the magnetic silicaparticles. The elution buffer (E) alters the pH of the magnetic silicaparticles in the eluted sample. In one embodiment, the tube containingthe eluted sample is transferred to another incubation/agitation ring 26by lowering the tube containing the eluted sample from the tube conveyor24 to the incubation/agitation ring 26 using a pneumatically-activatedelevator (not shown).

The incubation/agitation ring 26 shown in FIG. 2 includes twenty (20)positions to hold the tubes containing the eluted samples and is capableof rotation at a speed of about 1100 RPMs during incubation. Thoseskilled in the art can appreciate that the number of positions in theincubation/agitation ring 26 is a function of many things, includingdesired throughput and time. Therefore, use of a incubation/agitationring 26 having more or fewer positions than twenty is within the scopeand spirit of this disclosure.

The tubes containing the eluted samples are inserted and removedindividually. Once all twenty positions of the incubation/agitation ring26 have been filled with tubes, the tubes are incubated and agitatedcollectively as a group. Incubation and agitation occur for about ten(10) minutes at an incubation temperature of about 70° C. Once again,advantageously, unlike conventional batch processing, samples containinga different NA, such as HIV, HCV, HDB, and so forth, can be insertedrandomly into the incubation/agitation ring 26.

The altered pH, incubation temperature, and agitation promotedissociation of the NA from the magnetic silica particles andre-suspension of the NA in the solution of the eluted sample. The elutedsolution, which is an aqueous solution including the elution buffer andthe NA-enriched sample in suspension, can then be separated from thesolid, magnetic silica particles, e.g., by magnetic attraction andaspiration using a disposable pipette tip.

The eluted solution can be PCR tested immediately (as described below),or discrete tubes containing eluted solution can be capped andtemporarily stored for testing at some later time as described below ordiscrete tubes containing eluted solution and a Mastermix reagent can becapped and temporarily stored for testing at some later time also asdescribed below. The magnetic silica particles are disposed of.

In some applications, NA-purified, eluted samples may be desired fortesting at a later time and/or place. For such instances, the system 10includes a transfer point 31 after the wash cycle. More specifically,after washing and eluting, and separating the eluted solution from themagnetic silica particles, the tube containing the NA-purified, elutedsolution can be transported to a capping device 28, where the tubecontaining the NA-purified, eluted samples is sealed or capped tightly.After capping, the eluted solution can be tagged, e.g., by radiofrequency identification (RFID) using a radio “tagger” 33, and can thenbe stored in a refrigeration unit 35.

PCR Testing Apparatus

As provided in the description above, after separation of the magneticsilica particles from the eluted solution, the NA-purified, elutedsolution is recovered from the tube, e.g., using a pipettor having adisposable tip. Referring to FIG. 3, the recovered eluted solution isdispensed into a removable, reaction vessel or cuvette 40 (hereinafter“cuvette”) that is disposed temporarily on an incubation ring orconveyor belt 32. The cuvette 40 is structured and arranged to hold thereaction liquid (defined below) during at least one of capping andthermal cycling.

Preferably, the eluted solution is dispensed into a cuvette 40 thatcontains a Mastermix reagent, such as PCR MasterMix reagent manufacturedby Promega Corporation of Madison, Wis., (MM1 or MM2). Alternatively,the eluted solution can be added to the cuvette 40 first and theMastermix reagent can be added afterwards. The aqueous solutioncomprising Mastermix reagents and the eluted solution is referred to asa “reaction liquid”.

Mastermix reagents are well know to those skilled in the art and willnot be described in greater detail. The composition of the Mastermixreagent, each of which is prepared in advance and stored in a reagenttray 34, differs depending on the NA being tested for, such as HIV, HCV,HDB, and so forth. Each Mastermix reagent includes the reagenturacil-DNA-gylcosylase (UNG). UNG is a reagent that is inactive athigher temperatures; but, at lower temperatures, such as ambient or roomtemperature, UNG destroys any NA copies that may still be mixed in withthe reaction liquid. Thus, UNG is added to the reaction liquid tomitigate carryover contamination from the isolation/purificationapparatus 20 into the PCR testing apparatus 30.

Once Mastermix reagent and the eluted solution have been added to acuvette 40, a capping device 28 disposed along the incubation ring 32seals or caps each cuvette 40 tightly. As mentioned previously, not allsamples or, in this case, reaction liquids are prepared for immediatePCR testing. In some instances, it may be desirable to test discretereaction liquids at a later time or place. In such instances, discrete,capped, cuvettes 40 containing reaction liquids that have beenUNG-incubated, can be removed from the incubation ring 32 at a transferpoint 31 whence they can be transferred to a tagging device 33, such asan RFID radio “tagger”. The capped, cuvettes 40 so removed can be taggedand then stored in a refrigeration unit 35.

As shown in FIG. 3, in an illustrative embodiment, the incubation ring32 includes sixteen (16) positions to hold cuvettes 40 and the conveyingbelt is capable of advancing continuously at a rate so that each cuvette40 spends approximately ten (10) minutes processing along the incubationring 32. The temperature of the incubation ring 32 is maintained atabout 25° C.

Those skilled in the art can appreciate that the number of positions inthe incubation ring 32 is a function of many things, including desiredthroughput and time. Therefore, use of an incubation ring 32 having moreor fewer positions than sixteen is within the scope and spirit of thisdisclosure.

As the conveying belt of the incubation ring 32 continues to advance,capped, UNG-incubated cuvettes 40 containing reaction liquid are furtherincubated in an RT incubation portion 38, e.g., an oven, at atemperature of about 45° C. The purpose of incubating the cuvettes 40 ata temperature of about 45° C. is for converting any single-stranded RNAsamples into double-stranded DNA prior to PCR testing. Moreparticularly, the Mastermix reagent added to cuvettes 40 that containRNA samples instead of DNA samples will include reverse transcriptase(RT), a DNA polymerase enzyme that transcribes single-stranded RNA intodouble-stranded DNA.

As shown in FIG. 3, the RT incubation portion 38 includes fifty (50)positions to hold cuvettes 40 and the incubation ring 32 is capable ofadvancing incrementally or continuously at a rate so that each cuvette40 spends approximately thirty (30) minutes undergoing RT incubation.Those skilled in the art can appreciate that the number of positions inthe RT incubation portion 38 of the incubation ring 32 is a function ofmany things, including desired throughput and time. Therefore, use of anRT incubation portion 38 having more or fewer positions than 50 iswithin the scope and spirit of this disclosure.

The PCR testing apparatus 30 is further structured and arranged so thatthe point of termination of the RT incubation portion 38 is proximate toa PCR transfer point 37. As a result, at the completion of RTincubation, each capped, RT-incubated cuvette 40 is transferred from theincubation ring 32 to a denaturing heating station ring 39.

As shown in FIG. 3, the denaturing heating station ring 39 includessixteen (16) positions to hold cuvettes 40. The ring 39 is capable ofadvancing incrementally or continuously at a rate so that each cuvette40 spends approximately ten (10) minutes undergoing the denaturingthermal cycle. Those skilled in the art can appreciate that the numberof positions in the denaturing heating station ring 39 is a function ofmany things, including desired throughput and time. Therefore, use of adenaturing heating station ring 39 having more or fewer positions thansixteen is within the scope and spirit of this disclosure.

During the denaturing thermal cycle, each capped cuvette 40 is heated toa temperature between approximately 90 and 95° C., e.g., using an oven,to activate the polymerase enzyme (typically Taq) in the reactionliquid. As mentioned above, at approximately 90 and 95° C., the DNAdouble helix is melted so that each strand of the double helix isseparated from the other.

The capped cuvette 40 containing denatured NA is then transferred to anyvacant cuvette well 52 in an amplification and detection (A+D) module50. Preferably, the cuvettes 40 are transferred using a radial arm 55that is structured and arranged to pick-up the capped cuvette 40 fromthe denaturing heating station ring 39 and depositing it in a vacantcuvette well 52.

The A+D module 50 is a thermal cycler array having forty (40)individually-controllable, single-vessel thermal cyclers that arestructured and arranged to hold cuvettes 40 during the initial annealingand initial elongation thermal cycles and during all subsequent thermalcycles of the PCR process. Those skilled in the art can appreciate thatthe number of individually-controllable, single-vessel thermal cyclersin the A+D module 50 is a function of many things, including desiredthroughput and time. Therefore, use of an A+D module 50 having more orfewer individually-controllable, single-vessel thermal cyclers thanforty is within the scope and spirit of this disclosure.

While each capped cuvette 40 is disposed in a respective cuvette well52, the initial annealing and initial elongation thermal cycles of thePCR process are conducted at appropriate, pre-determined temperaturesranging between approximately 50 and 65° C. (for annealing) and betweenapproximately 70 and 72° C. (for elongation), respectively. After eachcomplete PCR cycle, the light intensity of each reaction liquid isdetected and measured. Once the light intensity reaches or exceeds apre-determined threshold intensity, I_(TH), the initial DNAconcentration of the NA sample can be estimated, for example usinglook-up tables.

Referring to FIG. 5, the A+D module 50 is in operational associationwith a fluorimeter 58 via a fiber-optic bundle 51. More specifically, atleast one optical fiber 53 of the fiber-optic bundle 51 is structuredand arranged to be optically-coupled to each cuvette well 52 and, moreparticularly, each cuvette 40 is optically-transmissive at requiredwavelengths. Each optical fiber 53 is optically-coupled to thefluorimeter 58 so that the intensity of light of reaction liquid can bemeasured at a plurality of wavelengths, such as four (4), following eachcomplete PCR cycle.

The fluorimeter 58 includes multiple, such as four (4), light sources(not shown) having different wavelengths or a single light source 54having a narrowband wavelength in combination with a filter wheel 56that can produce different wavelengths. When a single light 54 source isused, light from the fluorimeter 58 at a first wavelength illuminatesthe reaction liquid disposed in each cuvette well 52 via at least oneassociated optical fiber 53. The fluorescence signal from the reactionliquid is transmitted back to the fluorimeter 58 via at least oneassociated optical fiber 53. The fluorimeter 58 detects or measures theintensity level of the fluorescence signal. This process is repeated forat least three different wavelengths for each cuvette well 40 containinga reaction liquid.

The fluorimeter 58 also includes or is in operational association with aprocessing unit 57 and memory components 59 a and 59 b. Non-volatilememory 59 a can store applications or driver programs for operation ofthe fluorimeter 58, threshold intensity data, I_(TH), and/or look-uptables, LUT, correlating measured intensity level and number of PCRamplification cycles to estimate initial DNA concentration levels, andso forth. Memory component 59 b can include volatile, random accessmemory (RAM) for running one or more of the applications or driverprograms.

During operation of the system 10 and the PCR testing apparatus 30, theintensity of a detected fluorescence signal at a particular wavelength,I_(m), can be compared with a common, pre-determined thresholdintensity, I_(TH), applicable to all reaction liquids or with apre-determined threshold intensity, I_(TH), applicable to a specificreaction liquid. When the detected intensity level of the fluorescencesignal exceeds the threshold intensity (I_(m)>I_(TH)), look-up tables(LUT) can be used to correlate the initial NA concentration with themeasured intensity level, I_(m), wavelength, and number of amplificationcycles, n. The initial concentration for each reaction liquid(corresponding to an NA sample) can be recorded and saved in volatile ornon-volatile memory 59 b or 59 a.

Once initial concentration data for reaction liquid have been recorded,the cuvette 40 containing the reaction liquid can be removed from theA+D module 50 to await final disposition. Optionally, the cuvettes 40can be tagged by the RFID radio tagger 33 and stored in therefrigeration unit 35.

Single-Vessel Thermal Cycler

As provided above, the A+D module 50 is a circular, thermal cycler arrayhaving a plurality, e.g., forty (40), individually-controllable,single-vessel thermal cyclers. The single-vessel thermal cyclers arestructured and arranged to hold each cuvette 40 in a seat. Moreover,while each cuvette 40 is disposed in a seat in the A+D module 50, thetemperature of the cuvette 40 during the initial annealing and initialelongation thermal cycles and during all subsequent thermal cycles ofthe PCR process can be controlled individually. Individual thermalcontrol and the capability to continuously insert cuvettes 40 intoand/or extract cuvettes 40 from the A+D module 50 makes random accesstesting possible and practical.

Referring to FIG. 4, a valve-less, multi-vessel thermal cycler array 65having a plurality of single-vessel thermal cyclers 60 is shown.Although the invention is described using a valve-less approach,valve-based approaches for thermally-controlling the cuvettes 40 canalso be used without violating the scope and spirit of this disclosure.Advantageously, valve-based control systems may be quicker and may offermore precise temperature control. Disadvantageously, valve-based controlsystems are more expensive, more complex, and less reliable.

The multi-vessel thermal cycler array 65 includes a plurality of seats69 for accommodating or mounting radially-symmetrical cuvettes 40containing reaction liquid. The seats 69 are structured and arranged toprovide precise positioning of the cuvettes 40 with respect to therespective heating assembly (described below). Preferably, the seats 69are coaxial with a gas-to-vessel flow guide 67 (described below). Morepreferably, the seats 69 do not physically contact the gas-to-vesselflow guide 67 so as to provide an air gap 75 between the bottom portionof the cuvette 40 and the gas-to-vessel flow guide 67.

The seats 69 also may include a slight tilt in combination with aV-notch (not shown) or some other element or aspect as a centeringfeature. Optionally, the seats 69 can be structured and arranged tovibrate the reaction liquid 45 in the cuvette 40, for example, to reducetemperature gradients in the reaction liquid 45 and/or to out-gasbubbles that may obstruct optical viewing. For example, each vessel seat69 can be an electrically-actuated, AC-driven, piezoelectric disk havinga center hole region in which the cuvette 40 can sit.

To provide independent thermal control, each single-vessel thermalcycler 60 includes a heating assembly and a gas-to-vessel flow guide 67.The heating assembly includes a heating element 64, such as anelectrically-resistive heating element, which is disposed inside atubular, gas-to-heater flow guide 63. The gas-to-heater flow guide 63confines and distributes gas flow 68 relative to the heating element 64to achieve the proper heat transfer between the heating element 64 andthe gas 68.

The gas-to-vessel flow guide 67 acts as an outer shell of the “heatexchanger”. More specifically, the gas-to-vessel flow guide 67 is ashroud-like device that is structured and arranged to promotegas-to-vessel heat exchange by distributing the flow of gas 68 across aregion including the cuvette's 40 outer surface area 41 that correspondsto the reaction liquid-wetted, inner surface area 43. The gas-to-vesselflow guide 67 also includes an optically-transmissive region(s) 66 that,preferably, is/are in operational association with the optical-fiber(s)53 of the fiber-optics bundle 51 described above.

Gas 68, which can be a single gas or a mixture of gases, is the workingfluid for heat exchange with the cuvette 40. A gas flow driver 61, suchas an air compressor or diaphragm-type air pump, can be used to delivergas 68 through a main conduit 62 to each of the gas-to-vessel flowguides 67.

A controller 75 having a processing unit and volatile and non-volatilememories is electrically-coupled to the gas flow driver 61 as well as toeach of the heating elements 64. The controller 75 controls the rate ofgas 68 flow to the gas-to-vessel flow guides 67. The controller alsocontrols the timing and amount of current to each heating element 64 forthe purpose of controlling the heat exchange between the resistiveheating elements 64 and the gas 68 for each gas-to-vessel flow guide 67,independent of any other gas-to-vessel flow guide 67. Thus, at any pointin time, the temperature on any cuvette 40 is vessel specific andindependently variable.

Attainable air temperature change characteristics using, for example,5.4 liter/minute air flow and electrical heating of a coil of NiCr wireinclude a heating change rate of about 3.7° C./second from 60° C. to 90°C. and a cooling rate of about 2.5° C./second from 90° C. to 60° C.

The invention has been described in detail including the preferredembodiments thereof. However, those skilled in the art, upon consideringthe present disclosure, may make modifications and improvements withinthe spirit and scope of the invention.

What we claim is:
 1. A system comprising: a thermal cycler array havinga plurality of wells arranged in a circular array, each well arranged toaccommodate a single reaction vessel containing a reaction liquid, aplurality of resistive heating elements, each proximately located to adifferent one of the wells, and a plurality of flow guides, eacharranged to direct heat generated by one of the resistive heatingelements to only one of the wells; and a controller configured toindependently control the amount of current delivered to each resistiveheating element.
 2. The system of claim 1, further comprising: an armarranged to deliver a reaction vessel to one of the plurality of wells.3. The system of claim 2, further comprising: a denaturing heatingstation, from which the arm delivers the reaction vessel to one of theplurality of wells.
 4. The system of claim 3, wherein the denaturingheating station is arranged as a rotatable ring.
 5. The system of claim1, further comprising a fluorimeter in operational association with theplurality of wells via a plurality of optical fibers.
 6. The system ofclaim 5, wherein each flow guide includes an optically-transmissiveregion in operational association with at least one of the plurality ofoptical fibers.
 7. The system of claim 1, further comprising: a gassource; and a gas flow driver for providing pressurized gas from the gassource to each of the plurality of flow guides, wherein each of theresistive heating elements is arranged to variably heat the pressurizedgas before the pressurized gas reaches each respective flow guide. 8.The system of claim 1, wherein each of the plurality of wells isstructured and arranged to include at least one of a slight tilt, aV-notch, and a reaction vessel centering feature.